We continue to investigate the molecular basis of topographic phosphorylation of cytoskeletal proteins using squid giant axon system. Within the last decade a novel level of modulation of protein phosphorylation has emerged, namely, factors that regulate the conformation and stability of proteins phosphorylated at Ser/Thr-Pro sites by proline directed kinases. Most proline directed Ser/Thr kinases and phosphatases are highly selective for trans Ser/Thr-P bonds. Peptidyl-prolyl cis/trans isomerases such as Pin 1 specifically target phosphorylated Ser/Thr-P sites and by virtue of the proline residue, can toggle an unstable cis isomer to the more stable trans form, with altered function. Pin 1 plays a key role in diverse cellular functions, including the cell cycle, cancer, neurodegeneration, and apoptosis. Pin 1 is localized in nuclei of most cells, where it modulates the functions of several mitotic proteins. In neurons, however, Pin1 is distributed in both nucleus and cytoplasm, increases during neuronal differentiation and its expression correlates with the phosphorylation of tau at specific proline directed Ser/Thr residues and Pin1 is detected in neurofibrillary tangles in AD brains. Like tau, neurofilaments contain large number of proline directed Ser/Thr phosphate acceptor sites that are targeted by Pin1. In contrast to tau, however, NFs, particularly NF-M/H, are enriched with numerous KSP repeat motifs (43-100 depending on species) in the tail domain, sites for proline directed kinase phosphorylation. The question arises: Does Pin 1 play any role in stabilizing the numerous phosphorylated KSP sites in neurofilaments? Pin1 may play a role in regulating NF-H phosphorylation, particularly at the numerous KSP tail domain sites and may contribute to the neuronal pathology. To explore this question we resorted to GST-Pin 1 pull down assays of rat brain lysates. Coomassie stained gels and Western blots were prepared using an antibody specific for phosphorylated NF-H, (RT-97). A Coomassie band presumed to be NF-H was excised and identified mass spectrometrically as p-NF-H. Pin1 and p-NF-H also co-immunoprecipitate from rat brain lysates, linking Pin 1 to phosphorylated NF-H tail domain KSP repeats . The preliminary data suggest that Pin1 binds to pNF-H. The question is, what role does it play and how? As an initial hypothesis we suggest that Pin 1 is essential in stabilizing the phosphorylated KSP repeats in the tail domain as they are being phosphorylated by proline directed kinases during axonal transport. Several experiments are proposed to test this hypothesis using two compartments of giant axon system of squid, cell bodies and axon. Pure axoplasm free from cell body can be isolated from a single of the squid giant axon system the expression of Pin1 and the stable proline directed Ser/Thr phosphorylation of neurofilament proteins can be quantitated in both compartments. These studies will provide the role of Pin1 in stabilization of certain type of phosphorylation by a class of kinases known as proline directed Ser/Thr kinases. Pin1 regulates the extent of NF-H phosphorylation in vitro. Recombinant unphosphorylated rat NF-H can be expressed and purified . Erk1/2 can be obtained commercially while recombinant Pin1 will also be expressed and purified to set up an in vitro assay to determine the effect of Pin 1 on the phosphorylation of NF-H. Most KSP sites in rat NF-H are targeted by Erk1/2. To a constant amount of recombinant NF-H and Erk1/2, varying amounts of Pin1 will be added together with ATP and incubated for a constant time (2-3 hrs). The phosphorylation of NF-H will be analyzed by Western blot analysis using an antibody specific for p-NFH (RT-97, SMI31). The hypothesis predicts that with increasing amounts of Pin1, there should be a corresponding increase in the expression of stable p-NF-H. Finally, a specific inhibitor of Pin 1 activity, juglone, should inhibit in vitro NF-H phosphorylation should support the hypothesis. We test the following hypothesis; Hypothesis 1.1. Pin1 expression in a cell system will regulate the phosphorylation of NF-H HEK 293T cells are used routinely in our laboratory for transfection studies. Dr. S. Gutkind at the NICR, working on Pin 1 in cancer, has agreed to provide us with a Pin 1 construct for transfection. We shall also prepare a rat NF-H, or NF-M construct for cotransfection. HEK cells shall be doubly transfected with both constructs according to procedures established in our laboratory and the level of expression for both genes shall be determined in cell lysates by SDS-PAGE and Western blotting using Pin 1 monoclonal antibody and an antibody specific for total NF-H. Cells will be transfected with NF-H and Pin1, vector alone, Pin1 alone and with NF-H alone as controls. When treated with ectodermal growth factor (EGF) HEK cells show elevated Erk1/2 kinase activity. The hypothesis predicts that cells co-transfected with Pin 1 and NF-H will express high levels of p-NF-H compared to controls after EGF induction. Treatment of these cells with the Pin1 inhibitor, juglone, should inhibit p-NF-H expression. These studies will support the hypothesis. Pin 1 is associated with the perikaryal pathology seen in stressed neurons Neuronal stress (e.g.,oxidative, excitotoxic) deregulates the relative activities of kinases and phosphatases that tightly regulate topographic phosphorylation. This deregulation involves abnormal activation of proline directed kinases such as cdk5, Erk1/2, SAPK, and p38, and results in pNF-H (and/or hyperphosphorylated tau) accumulations within perikarya, leading to cell death (Davis et al., 1995; Brownlees et al., 2000; Shea et al., 2004). Since Pin1 is expressed in neuronal cell nuclei and cytoplasm, stress upregulation of proline directed kinases within cell bodies, may also evoke a Pin1 response and stimulate aberrant NF-H phosphorylation. To test this, we plan to use primary rat dorsal root ganglion ( DRG) neurons (E16-18) in culture for several days challenged with an excitotoxic stress stimulus (glutamate treatment) which is known to induce perikaryal phospho-tau and NF accumulations and cell death. After treatment, some cultures will be prepared as lysates while others will be fixed for immunofluorescent ICC assays to localize Pin1 and p-NF-H. If activation of proline directed kinases occurs, then we would predict that the levels of p-NFH would be significantly increased in the stressed neurons compared to controls. In addition, we predict that ICC assays should show that Pin1 and p-NFH are co-localized in perikarya of stressed neurons, resembling the pathology in ALS motor neurons. Both effects should be rescued by prior treatment of cells with juglone, the Pin1 specific inhibitor. Although Pin is expressed in cell bodies, these results would suggest that stress induced elevation of proline directed kinases in the cell body is primarily responsible for aberrant phosphorylation of KSP sites while Pin1 acts to stabilize them as they are formed.